Competitive binding analysis by fluorescence

Abstract

Thyroid hormones such as thyroxine and triiodothyronine are assayed in biological fluid samples by a competitive binding analysis in which a fluorescent label is used. Upon formation of complexes, the fluorescence of the labelled compound is released, and the hormone can be assayed by measuring the fluorescence of the mixture and comparing the result with standard data. Fluorescein is the preferred label. The process can be operated in a continuous flow manner.

What is claimed is: 
     
       1. A method of assaying a biological fluid sample for a thyroid hormone, comprising the steps of: (a) forming a mixture of said fluid sample with a known amount of conjugate formed by labeling a thyroid hormone with a fluorophore, said fluorophore having a fluorescent level which is substantially suppressed by said thyroid hormone in said conjugate;   introducing into said mixture a binding substance which will equilibrate with both the thyroid hormone in said conjugate and said fluid sample, said binding substance sterically hindering the thyroid hormone of said conjugate from suppressing the fluorescence of said fluorophore;   (c) measuring the fluorescent level of said mixture; and   (d) calculating the amount of thyroid hormone in said sample by comparing the fluorescent level of said mixture with a standard fluorescent level.   
     
     
       2. A method according to claim 1 wherein the biological fluid sample contains a thyroid hormone selected from a group consisting of thyroxine and triodothyronine. 
     
     
       3. A method according to claim 1, wherein the fluorophore from which the said conjugate has been formed is fluorescein. 
     
     
       4. A method according to claim 1, wherein the fluorophore from which the said conjugate has been formed is selected from a group consisting of: dansyl, rhodamine, fluorescamine, pyrene and 2-methoxy-2,4-dyphenyl-3(2H)-furanone. 
     
     
       5. A method of assaying a biological fluid sample for thyroxine which comprises the steps of: (1) forming a mixture of a sample containing an unknown amount of thyroxine with (a) a known amount of fluorescein-labelled thyroxine; and   (b) anti-thyroxine antiserum; (2) measuring the fluorescence of the mixture so formed; and (3) calculating the amount of thyroxine contained in the sample from the fluorescence measurement as compared with standard fluorescent data.   
     
     
       6. A method of assaying a sample fluid for a thyroid hormone, which contains iodine, comprising (a) forming a mixture of said sample with a known amount of a conjugate formed by labeling said thyroid hormone with a fluorophore;   (b) adding to said mixture a known amount of a binding substance for said thyroid hormone, said binding substance hindering the fluorescence inhibiting action of the iodine contained in said thyroid hormone;   (c) measuring the fluorescent level of the mixture;   (d) calculating the amount of thyroid hormone in said sample by comparing the fluorescent level with that of a standard.